Dickkopf-1在骨质调节中的研究新进展

Dickkopf-1(DKK-1)是Wnt途径中的一个可溶性抑制物,在骨质形成与破坏等方面有着积极的作用[1].Wnt信号通路广泛存在于无脊柱动物和脊柱动物中,是一类在物种进化过程中高度保守的信号通路.早期即有Wnt信号在调节生长发育、器官形成等重要生理功能方面的相关报道[2],在骨生理方面的作用直到最近才被报道[3].Wnt信号通路是一个复杂的信号调控网络,目前认为至少存在4条Wnt信号通道:①Wnt/β-连环蛋白(β-catenin)途径:亦称经典信号通路,通过激活β-catenin基因转录;②Wnt/平面细胞极性(planar cell polarity,PCP)途径:通过小G蛋白激活c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)来调控细胞功能;③Wnt/Ca2+途径:通过释放细胞内Ca2+来调控相关功能;④Wnt/蛋白激酶A途径:通过激活蛋白激酶A调控细胞功能.其中Wnt/β-catenin途径是最重要的一条途径,在关节及骨生理等方面有重要调控作用[4].DKK-1是Wnt途径重要的调控分子之一,是关节重构的关键调控者.在关节炎动物实验中,高水平的DKK-1被认为与骨质吸收的模型相关;相反,低水平的DKK-1与新骨形成的模型相关[5].本文主要探讨DKK-1在关节重构以及骨质调节方面的研究新进展.     

骨性关节炎软骨和软骨下骨之间信号通路

骨性关节炎(osteoarthritis,OA)的发生和发展离不开软骨和软骨下骨共同病变的过程。信号通路的异常在调控OA软骨下骨和软骨的病变中起重要作用。Wnt、转化生长因子β(transforming growth factorβ,TGFβ)/骨形态发生蛋白(bone morphogenetic protein,BMP)、丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)信号通路对骨和软骨正常生长发育和代谢有着重要的调控作用,维持了关节的健康和平衡。研究显示,在OA中,这些信号通路的改变不仅可使OA软骨下骨和软骨的细胞表型和分子功能失衡,细胞外基质的合成破坏,软骨下骨骨重塑,还可通过破坏组织细胞的代谢进一步改变骨和软骨的结构及应力承担能力。因此,本文围绕骨关节炎病变中Wnt、TGFβ/BMP、MAPK信号交流在OA中软骨下骨和软骨病变中的作用和机制进行综述,以期为OA和其他骨关节疾病的研究和治疗提供新的方法和思路。     

Wnt信号通路与胰岛β细胞增殖的关系

Wnt信号转导通路参与调节细胞的增殖、分化、运动及凋亡等,是细胞发育及形态形成所必需.近年来研究发现Wnt信号通路的卷曲蛋白受体在人的胚胎以及成年胰腺的外分泌部和胰岛细胞上都有表达,Wnt信号通路的各种组成成分与胰岛β细胞增殖及胰岛素分泌关系密切.此外,Wnt信号通路的下游靶基因可以调控细胞周期的进程,提示Wnt信号通路在胰岛β细胞增殖、分化过程中发挥重要的作用.     

β连环蛋白翻译后修饰与肾间质纤维化

β连环蛋白(β-catenin)在细胞中具有双重作用,一是参与钙黏蛋白(cadherin)介导的细胞间的黏附作用,二是作为经典wnt信号通路中最重要的信息分子,调控细胞生长、分化和凋亡等。β-catenin能发生多种蛋白翻译后修饰,如磷酸化、泛素化、乙酰化,影响其自身稳定性、细胞定位及活性,进而介导E-cadherin/β-catenin复合物完整性,并调节Wnt/β-catenin信号通路,调控β-catenin靶基因的表达,最终影响肾间质纤维化的发生发展过程。     

miR-183调控Wnt/β-catenin信号通路影响胃癌SGC-7901细胞生物学特性的研究

背景 miR-183在胃癌、乳腺癌、膀胱癌等多种肿瘤组织中低表达,发挥抑癌基因的作用,但其在胃癌中作用机制目前尚不十分清楚.研究表明, miR-183可以通过调节Wnt/β-catenin信号通路抑制骨肉瘤细胞的生长、迁移和侵袭,而Wnt/β-catenin信号通路在胃癌中高度激活与胃癌的发生和转移密切相关.但miR-183是否调节Wnt/β-catenin信号通路影响胃癌细胞生物学特性尚不清楚.目的探讨miR-183调控Wnt/β-catenin信号通路对胃癌细胞生物学特性的影响.方法采用q RT-PCR检测miR-183在不同胃癌细胞株中的表达情况,在胃癌细胞SGC-7901中转染miR-183mimics或mimics对照,分别设为miR-183组和miR-NC组, qRT-PCR检测转染效率,噻唑蓝增殖实验检测SGC-7901细胞增殖变化,流式细胞仪检测SGC-7901细胞凋亡情况, Transwell实验检测SGC-7901细胞侵袭和迁移能力, Western blot法检测凋亡相关及Wnt/β-catenin信号通路相关蛋白表达水平.使用Wnt/β-catenin信号通路激动剂氯化锂处理过表达miR-183的SGC-7901细胞,观察SGC-7901细胞生物学特性的变化.结果与正常胃黏膜上皮GES-1细胞相比, 4株胃癌细胞中miR-183的表达水平明显降低(P 0.05).转染miR-183mimics后SGC-7901细胞中miR-183的表达水平显著升高(P0.05).过表达miR-183后SGC-7901细胞OD值降低(P0.05),凋亡率、Bax和Cleaved Caspase-3蛋白表达水平升高(P 0.05),侵袭和迁移细胞数减少(P0.05),β-catenin、p-GSK-3β和Cyclin D1蛋白表达水平下调(P0.05), GSK-3β蛋白表达水平上调(P 0.05).激活Wnt/β-catenin信号通路部分逆转了过表达miR-183对SGC-7901细胞增殖、侵袭和迁移的抑制作用及凋亡促进作用(P0.05).结论miR-183可能通过抑制Wnt/β-catenin信号通路阻碍人胃癌SGC-7901细胞增殖、侵袭和迁移能力,促进细胞凋亡.     

骨形态发生蛋白和Wnt信号通路在心脏发育及生物起搏中的研究进展

心脏胚胎发育过程由骨形态发生蛋白(BMP)、Wnt、Notch等信号通路共同调控,其中BMP和Wnt信号通路是心脏胚胎发育过程中两条重要的信号通路。BMP通过由转化生长因子-β超家族转导的Smad和TAK1两条信号途径诱导胚胎干细胞向心肌细胞分化。Wnt信号通路对胚胎发育、细胞极性决定、细胞特异性分化、心血管系统发育具有重要的调控作用,在心脏胚胎发育早期促进中胚层的形成,在后期抑制其向心肌细胞分化;但这两条信号通路共同诱导干细胞向心肌细胞分化的效率高于BMP或Wnt信号通路的诱导效率。BMP和Wnt信号通路共同诱导干细胞分化的心肌细胞,不仅为药物实验、心脏疾病研究提供体外模型,而且也为构建生物起搏器提供重要基础。     

自噬相关信号通路调控骨相关疾病发生发展的作用机制研究进展

自噬是细胞内大分子物质与细胞器被次级溶酶体进行降解与消化的过程，借此维持细胞的自我稳定。自噬在维持骨骼的动态平衡中具有关键性的调控作用，其可通过磷酸酰肌醇3激酶（PI3K）/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白（mTOR）、核因子κB(NF-κB)、Wnt/β-连环蛋白（β-catenin）、腺苷单磷酸激活的蛋白激酶（AMPK）等信号通路调控成骨细胞、软骨细胞、骨髓间充质干细胞和破骨细胞的增殖、分化等生物学行为，调控骨相关疾病的发生发展。     

Wnt-3a对Connexin43及相关基因的表达调控

目的 探讨信号分子Wnt3a蛋白对缝隙连接蛋白43(connexin43)的表达调控作用,以期揭示Wnt3a与connexin43在心脏发育过程中的特定联系与作用.方法 以本室自行构建pCD-NA3.1/wnt-3a真核表达载体转染大鼠心肌细胞系H9c2,在细胞水平通过RT-PCR及Western-blot实验方法研究Wnt-3a对其中Cx43表达及心脏发育相关基因的影响.结果 经转染wnt-3a后,H9c2细胞系中connexin43的表达明显上调,同时与心脏发育相关的GATA4,Nkx2.5基因表达也明显上调.结论 Wnt3a信号通路对connexin43表达存在影响;并可能对心脏发育存在调控作用.     

RET指样蛋白3在恶性肿瘤发生发展中的作用研究进展

RET指样蛋白（RFPL）作为一类RET指样蛋白类似物家族，在多种恶性肿瘤中广泛表达。RET指样蛋白3(RFPL-3)是RFPL中的一员，在肿瘤的发生发展中发挥重要作用。在乳腺癌中，RFPL-3主要通过促进人端粒逆转录酶基因（hTERT）转录和上调端粒酶活性，来介导乳腺癌的发生。RFPL-3还可以通过MAPK通路和转化生长因子β(TGF-β)/Smad通路以及与p53重要的负调控因子双微体同源基因2(MDM2)竞争p53结合位点，从而促进肺癌的进展。而在睾丸生殖细胞肿瘤中，RFPL-3表达下调促进了肿瘤细胞的转移和增殖相关通路激活，包括细胞外基质—受体相互作用、黏着斑信号通路、黏附连接信号通路以及PI3K/Akt信号通路、Wnt信号通路和Hippo信号通路。在甲状腺癌的发生发展中，促进肿瘤细胞增殖的重要原因在于RFPL-3对Yes-associated蛋白的修饰，从而激活Hippo信号通路。因此，RFPL-3与多种恶性肿瘤的发生发展密切相关，有望成为治疗恶性肿瘤的潜在靶点，为患者的治疗提供更多选择。     

糖原合酶激酶3β信号通路在脑缺血中的作用

糖原合酶激酶3β( glycogen synthase kinase 3β,GSK3β)是一种丝氨酸/苏氨酸蛋白激酶.生长因子、Wnt等多条信号通路可抑制GSK3β活性,从而促进细胞存活.脑缺血时,随着GSK3β磷酸化水平发生改变,其上游和下游蛋白磷酸化水平也发生改变.缺血预处理和后处理可能通过调节GSK3β信号通路诱导脑缺血耐受.     

Ethanol affects differentiation-related pathways and suppresses Wnt signaling protein expression in human neural stem cells

Background: Prenatal exposure of the fetus to ethanol (EtOH) can be teratogenic. We previously showed that EtOH alters the cell fate of human neural stem cells (NSC). As Wnt signaling plays an important role in fetal brain development, we hypothesized that EtOH suppresses Wnt signaling protein expression in differentiating NSC and thereby contributes to fetal alcohol spectrum disorder. Methods: NSC isolated from fetal human brains were cultured in mitogenic media to induce neurospheres, which were dissociated into single‐cell suspensions and used for all experiments. Equal numbers of NSC were cultured on lysine/laminin‐coated plates for 96 hours in differentiating media containing 0, 20, or 100 mM EtOH. Total mRNA was isolated from samples containing 0 or 100 mM EtOH and changes in expression of 263 genes associated with neurogenesis and NSC differentiation were determined by Oligo GEArray technology. The biological impact of gene changes was estimated using a systems biology approach with pathway express software and KEGG database. Based on the pathways identified, expression of Wnt proteins (Wnt3a and Wnt5a), Wnt‐receptor complex proteins (p‐LRP6, LRP6, DVL2, and DVL3), Wnt antagonist Naked‐2 (NKD‐2), and downstream Wnt proteins (β‐catenin, Tyr‐p‐GSK3β, Ser‐p‐GSK3β) were analyzed by Western blot. Results: Of the 263 genes examined, the expressions of 22 genes in differentiating NSC were either upwardly or downwardly affected by EtOH. These genes are associated with 5 pathways/cellular processes: axon guidance; hedgehog signaling; TGF‐β signaling; cell adhesion molecules; and Wnt signaling. When compared to controls, EtOH, at both 20 and 100 mM concentrations, suppressed the expression of Wnt3a and Wnt5a, receptor complex proteins p‐LRP6, LRP6 and DVL2, and cytoplasmic proteins Ser‐p‐GSK3β and β‐catenin. Expression of NKD‐2 and DVL3 remained unchanged and the expression of active Tyr‐p‐GSK3β increased significantly. Conclusions: EtOH can significantly alter neural differentiation pathway‐related gene expression and suppress Wnt signaling proteins in differentiating human NSC.     

Inhibition of tumorigenesis driven by different Wnt proteins requires blockade of distinct ligand-binding regions by LRP6 antibodies

Disregulated Wnt/β-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer.     

Tissue-specific roles of Axin2 in the inhibition and activation of Wnt signaling in the mouse embryo

Axin proteins are key negative regulators of the canonical Wnt signal transduction pathway. Although Axin2 null mice are viable, we identified an unusual ENU-induced recessive allele of Axin2, canp, that causes midgestation lethality in homozygotes. We show that the Axin2(canp) mutation is a V26D substitution in an invariant N-terminal sequence motif and that the Axin2(canp) protein is more stable than wild type. As predicted for an increased level of a negative regulator, the Axin2(canp) mutation leads to decreased Wnt signaling in most tissues, and this can account for most of the morphological phenotypes of Axin2(canp) mutants. In contrast, there is a paradoxical increase in canonical Wnt activity in the late primitive streak of all Axin2(canp) mutant embryos that is associated with the formation of an ectopic tail in some mutants. Treatment of wild-type embryos with an inhibitor of Tankyrase that stabilizes Axin proteins also causes inhibition of Wnt signaling in anterior regions of the embryo and a gain of Wnt signaling in the primitive streak. The results indicate that although increased stability of Axin2 leads to a loss of canonical Wnt signaling in most tissues, stabilized Axin2 enhances Wnt pathway activity in a specific progenitor population in the late primitive streak.     

Wnt7a signaling promotes dendritic spine growth and synaptic strength through Ca²⁺/Calmodulin-dependent protein kinase II

The balance between excitatory and inhibitory synapses is crucial for normal brain function. Wnt proteins stimulate synapse formation by increasing synaptic assembly. However, it is unclear whether Wnt signaling differentially regulates the formation of excitatory and inhibitory synapses. Here, we demonstrate that Wnt7a preferentially stimulates excitatory synapse formation and function. In hippocampal neurons, Wnt7a increases the number of excitatory synapses, whereas inhibitory synapses are unaffected. Wnt7a or postsynaptic expression of Dishevelled-1 (Dvl1), a core Wnt signaling component, increases the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs), but not miniature inhibitory postsynaptic currents (mIPSCs). Wnt7a increases the density and maturity of dendritic spines, whereas Wnt7a-Dvl1-deficient mice exhibit defects in spine morphogenesis and mossy fiber-CA3 synaptic transmission in the hippocampus. Using a postsynaptic reporter for Ca(2+)/Calmodulin-dependent protein kinase II (CaMKII) activity, we demonstrate that Wnt7a rapidly activates CaMKII in spines. Importantly, CaMKII inhibition abolishes the effects of Wnt7a on spine growth and excitatory synaptic strength. These data indicate that Wnt7a signaling is critical to regulate spine growth and synaptic strength through the local activation of CaMKII at dendritic spines. Therefore, aberrant Wnt7a signaling may contribute to neurological disorders in which excitatory signaling is disrupted.     

Wnt/β-catenin signaling is differentially regulated by Gα proteins and contributes to fibrous dysplasia

Skeletal dysplasias are common disabling disorders characterized by aberrant growth of bone and cartilage leading to abnormal skeletal structures and functions, often attributable to defects in skeletal progenitor cells. The underlying molecular and cellular mechanisms of most skeletal dysplasias remain elusive. Although the Wnt/β-catenin signaling pathway is required for skeletal progenitor cells to differentiate along the osteoblastic lineage, inappropriately elevated levels of signaling can also inhibit bone formation by suppressing osteoblast maturation. Here, we investigate interactions of the four major Gα protein families (Gα(s), Gα(i/o), Gα(q/11), and Gα(12/13)) with the Wnt/β-catenin signaling pathway and identify a causative role of Wnt/β-catenin signaling in fibrous dysplasia (FD) of bone, a disease that exhibits abnormal differentiation of skeletal progenitor cells. The activating Gα(s) mutations that cause FD potentiated Wnt/β-catenin signaling, and removal of Gα(s) led to reduced Wnt/β-catenin signaling and decreased bone formation. We further show that activation of Wnt/β-catenin signaling in osteoblast progenitors results in an FD-like phenotype and reduction of β-catenin levels rescued differentiation defects of FD patient-derived stromal cells. Gα proteins may act at the level of β-catenin destruction complex assembly by binding Axin. Our results indicate that activated Gα proteins differentially regulate Wnt/β-catenin signaling but, importantly, are not required core components of Wnt/β-catenin signaling. Our data suggest that activated Gα proteins are playing physiologically significant roles during both skeletal development and disease by modulating Wnt/β-catenin signaling strength.